Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.
Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.
Haigler
B E
BE
Center for Applied Microbiology, University of Texas, Austin 78712.
Gibson
D T
DT
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D049308:Dioxygenases; D005288:Ferredoxins / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D010105:Oxygenases / Q000032:analysis*; D011549:Pseudomonas / Q000201:enzymology*
D000596:Amino Acids; D005288:Ferredoxins; D005415:Flavins; D009097:Multienzyme Complexes; D010088:Oxidoreductases; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase; C028878:ferredoxin-NAD+ reductase